Elsevier

Neuroscience

Volume 463, 21 May 2021, Pages 337-353
Neuroscience

Research Article
Early Hypoexcitability in a Subgroup of Spinal Motoneurons in Superoxide Dismutase 1 Transgenic Mice, a Model of Amyotrophic Lateral Sclerosis

https://doi.org/10.1016/j.neuroscience.2021.01.039Get rights and content

Highlights

  • Dendritic overbranching in lumbar motoneurons is common to low expressor lines of transgenic mice (SOD1G93A-low and SOD1G85R).

  • This overbranching occurs during the postnatal period in the “sustained firing pattern” subpopulation of lumbar motoneurons.

  • Main changes in electrical properties were found in the “delayed onset firing pattern” subpopulation of motoneurons.

  • The initial hypoexcitability detected in lumbar motoneurons might be the earliest pathological sign observed in SOD1 mice.

  • This dysfunction may lead to the late vulnerability of the future fast fatigable motoneurons.

Abstract

In amyotrophic lateral sclerosis (ALS), large motoneurons degenerate first, causing muscle weakness. Transgenic mouse models with a mutation in the gene encoding the enzyme superoxide dismutase 1 (SOD1) revealed that motoneurons innervating the fast-fatigable muscular fibres disconnect very early. The cause of this peripheric disconnection has not yet been established. Early pathological signs were described in motoneurons during the postnatal period of SOD1 transgenic mice. Here, we investigated whether the early changes of electrical and morphological properties previously reported in the SOD1G85R strain also occur in the SOD1G93A-low expressor line with particular attention to the different subsets of motoneurons defined by their discharge firing pattern (transient, sustained, or delayed-onset firing). Intracellular staining and recording were performed in lumbar motoneurons from entire brainstem-spinal cord preparations of SOD1G93A-low transgenic mice and their WT littermates during the second postnatal week. Our results show that SOD1G93A-low motoneurons exhibit a dendritic overbranching similar to that described previously in the SOD1G85R strain at the same age. Further we found an hypoexcitability in the delayed-onset firing SOD1G93A-low motoneurons (lower gain and higher voltage threshold). We conclude that dendritic overbranching and early hypoexcitability are common features of both low expressor SOD1 mutants (G85R and G93A-low). In the high-expressor SOD1G93A line, we found hyperexcitability in the sustained firing motoneurons at the same period, suggesting a delay in compensatory mechanisms. Overall, our results suggest that the hypoexcitability indicate an early dysfunction of the delayed-onset motoneurons and could account as early pathological signs of the disease.

Introduction

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease that affects cortical neurons in the motor cortex as well as spinal and brainstem motoneurons, except for those innervating the extraocular and pelvic muscles (Kanning et al., 2010, Robberecht and Philips, 2013). The discovery of the superoxide dismutase 1 (SOD1) mutation responsible for ALS (Rosen et al., 1993) led to the genetic engineering of transgenic SOD1 mice (Gurney et al., 1994, Bruijn et al., 1997, Bruijn et al., 1998), which stand today as the standard animal model of ALS by mimicking most of the pathological mechanisms leading to motoneuron degeneration. A SOD1-dependent pathogenic mechanism, common to familial and sporadic ALS, has been identified as wild-type (WT) SOD1 acquired toxicity following protein oxidation and conformational changes (Bosco et al., 2010). The early pathological signs appearing before the onset of clinical symptoms include slowed axonal transport (Williamson and Cleveland, 1999), altered excitability (Pieri et al., 2003, Kuo et al., 2005, Bories et al., 2007, Van Zundert et al., 2008, Van Zundert et al., 2012, Pambo-Pambo et al., 2009, Elbasiouny et al., 2010, Quinlan et al., 2011), denervation of axonal fibres (Pun et al., 2006, Hegedus et al., 2007, Saxena et al., 2009), neurofilament perturbations, mitochondrial alterations and glutamate excitotoxicity (Robberecht and Philips, 2013, Taylor et al., 2016). Unfortunately, no curative therapy is yet available, possibly due to late diagnosis (Robberecht and Philips, 2013). It has long been known that large spinal motoneurons are the first affected during the course of the disease (Kanning et al., 2010, Vucic et al., 2014, Taylor et al., 2016). Specifically, the fast-fatigable (FF) motoneurons disconnect in the SOD1 mouse model well before clinical symptoms (Pun et al., 2006, Hegedus et al., 2008, Saxena et al., 2009, Vinsant et al., 2013, Hadzipasic et al., 2014). However, the reasons for this peripheral nerve-muscle disjunction remain unknown.

We previously reported hypoexcitable lumbar motoneurons in SOD1G85R mice and dendritic overbranching occurring during the postnatal period (between postnatal days P3 and P8) (Durand et al., 2006, Amendola et al., 2007, Bories et al., 2007, Amendola and Durand, 2008, Filipchuk and Durand, 2012). Next, we addressed the cause of this abnormal dendritic growth in the SOD1G85R mice, and whether another strain of SOD1 mice (SOD1G93A-low) develops such dendritic overbranching and hypoexcitability at the same period. In our previous investigations in postnatal SOD1 mice, we found that the spinal networks involved in locomotor function were not responsive to conventional pharmacological agents (Amendola et al., 2004, Durand et al., 2006), and hypothesized that a delay descending pathways arrival could explain this dysfunction (Amendola et al., 2004, Durand et al., 2006). Such a delay would imply a reduced number of presynaptic afferents to motoneurons. In the present study we performed immunohistochemistry for synaptophysin in order to determine whether terminal boutons are missing in the region of motoneuronal pools in SOD1G85R mice.

Dendritic overbranching, intracellular recordings and neurobiotin staining allowed us to identify and compare the morphologies of lumbar motoneurons in SOD1G93A-low mice and their WT littermates. Recently, we classified lumbar motoneurons in postnatal mice into three subgroups according to their firing patterns (transient, sustained, and delayed-onset firing) (Durand et al., 2015). Transient motoneurons fire one or a few spikes during a long current pulse and disappear with maturation (Vinay et al., 2000, Mentis et al., 2007, Durand et al., 2015), whereas delayed-onset firing motoneurons exhibit a late spiking and display the highest rheobase compared to the transient and sustained firing types (Pambo-Pambo et al., 2009, Leroy et al., 2014, Durand et al., 2015). Sustained (or immediate) firing motoneurons discharge regularly throughout the entire current pulse and represent the predominant group after P9 (Durand et al., 2015). The present work aimed to determine whether sustained or delayed-onset firing subgroups of SOD1 motoneurons are preferentially affected during this early period (Durand et al., 2015). For comparison with previous studies on SOD1G85R mice (Amendola et al., 2007, Bories et al., 2007, Filipchuk and Durand, 2012, Durand et al., 2015), we targeted the same pools of lumbar SOD1G93A-low motoneurons that send their axons into the fifth ventral roots (see McHanwell and Biscoe, 1981). Finally, we focused on the electrical properties of lumbar motoneurons in the G93A high-expressor mutants, as no electrophysiological studies were yet performed in the entire brainstem-spinal cord preparation of the SOD1G93A-high strain. Thus, we could for the first time compare the electrical properties of the lumbar motoneurons in the low- and high-expressor lines at the same age and in the “en bloc” preparation.

Section snippets

Experimental procedures

All surgical and experimental procedures were in conformity with the European Communities Council directive (86/609/EEC) and the ethic committee of the Institut de Neurosciences de la Timone (n°71).

Results

Intracellular recordings of 78 antidromically identified lumbar motoneurons were performed in in vitro brainstem-spinal cord preparations from SOD1G93A-low (N = 11) and SOD1G93A-high (N = 10) mice and their respective non-transgenic littermates (N = 19 and N = 8), for a total of 45 WT and 33 SOD1G93A motoneurons. Among them, intracellular staining and full 3D complete reconstructions were obtained for 15 motoneurons of 15 mice (11 WT and 4 SOD1G93A-low). Immunohistochemistry were performed in 4

Discussion

In the present work, our results clearly demonstrate dendritic overbranching in SOD1G93A-low lumbar motoneurons compared to their non-transgenic WT littermates. Further we found an hypoexcitability in the delayed-onset firing group of motoneurons in the SOD1G93A-low mice suggesting early precursor sign of the disease. Our quantitative analysis of their dendritic morphologies shows that overbranching is a common feature of both SOD1G93A-low motoneurons (this work) and low-expressor SOD1G85R at

Acknowledgements

This work was supported by CNRS, Aix Marseille Université, France, grant n° 11890 from Association Française contre les Myopathies (AFM), Thierry Latran Foundation (OHEX Project) and Association pour la recherche sur la sclérose latérale amyotrophique et autres maladies du motoneurone (ARSLA), France. APP is a grant recipient from Government of Gabon. AF received a grant from the Ministère des Affaires Étrangères. The authors wish to thank Anne Duhoux for taking care of the animals.

Conflict of interest statement

The authors declare no conflict of interest.

Authors contribution

AF and APP contributed equally to this work, collecting and analyzing the experimental data. AF, APP JPG and JD performed most of the electrophysiological data analysis, and AF performed all the morphological analysis including 3D reconstructions with Neurolucida. FG and JD performed the electrophysiological experiments on the high expressor line SOD1G93A. SL and CB did immunohistochemistry. JD and JPG designed the research. All authors discussed the results and contributed to the manuscript.

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